The hepatitis C virus (HCV) is an important pathogen that has infected approximately 170 million people worldwide. In spite of extensive efforts, surprisingly little is known about the mechanism by which HCV initiates infection. The lack of vaccine or effective therapy against this virus stresses the urgent need for studies of HCV entry into host cells. HCV is an enveloped virus that enters cells via a clathrin-mediated pathway and releases its nucleocapsid by fusing the viral membrane with an endosomal membrane a process that is promoted by HCV E1 and E2 glycoproteins. The fusion induced by E1E2 is strictly receptor- and low pH-dependent. Not only is the mechanism of this process poorly understood, but even the identity of the fusion protein (E1 vs. E2) has not been established. This is due to the lack of a tractable model system to study HCV fusion. Our working hypothesis is that HCV fusion precedes through at least two major steps - a priming step through interactions with cellular receptors which render E1E2 fusogenic, followed by low pH-dependent fusion. This hypothesis predicts that: (1) low pH pre-treatment in the absence of receptors does not induce irreversible conformational changes in E1E2 leading to virus inactivation;(2) binding to soluble or to membrane-anchored receptors renders E1E2 competent to undergo conformational changes and promote fusion at low pH. These predictions will be tested using novel virus inactivation, virus-cell fusion and cell-cell fusion assays. Time-resolved imaging of single virus will be implemented for tracking HCV uptake and subsequent fusion with an endosome. HCV entry will be further examined by capturing and characterizing distinct intermediate stages of fusion. These experiments should provide new insights into the mechanism of E1E2-induced fusion and might help to design preventive strategies to block HCV infection. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is an important pathogen that has infected over 170 million people worldwide. Following HCV internalization by a cell, infection is initiated by merging of viral and cellular membranes that releases the viral genome. To elucidate the mechanism of membrane merger mediated by HCV glycoproteins, single virus entry into a host cell will be visualized by real-time by fluorescence microscopy. Understanding the virus entry process will suggest new strategies to prevent infection.